The acyl-CoA dehydrogenases (ACDs) are a family of multimeric flavoenzymes that catalyze the 1,2- dehydrogenation of acyl-CoA esters in fatty acid 2-oxidation and amino acid catabolism. Inborn errors of metabolism have been identified in seven of the ACDs. The long range objective of this project has been to investigate important structure/function relationships in the ACD gene family. We have described and characterized several new members of the ACD gene family. Among these are 3 enzymes with significant activities with long chain substrates: long and very long chain acyl-CoA dehydrogenases (LCAD and VLCAD, respectively), and ACD9 and. Our prior and preliminary studies show that these enzymes have distinct substrate utilization profiles, tissue and developmental expression patterns, exist in multiple active forms in the cell, and are present in multiple subcellular locations. The goal of this revised application is to characterize the physiologic roles of LCAD, VLCAD, and ACD9 and explore the ramifications of genetic deficiencies of these enzymes in humans and mouse models. Specific Aim 1 is to characterize variant forms of very long chain acyl-CoA dehydrogenase (VLCAD) and the molecular basis of clinical variability in this disorder. Specific aim 1a is to identify the amino acid motif(s) important in determining the unique localization of VLCAD to the inner mitochondrial membrane. Specific aim 1b is to characterize alternative forms of VLCAD identified in vivo. We have identified 3 variant forms of this enzyme in vivo that are generated through alternative splicing. I hypothesize that each has a different substrate specificity that provides functional optimization for progressively shorter substrate species. Specific aim 1c is to characterize the effect of patient mutations in VLCAD on enzyme function. Specific Aim 2 is to more completely characterize ACD9 and its deficiency in humans. Specific Aim 2a is identification of additional patients with ACD9 deficiency and definition of its clinical spectrum. Specific Aim 2b is characterization of the subcellular distribution of ACD9 and the function and molecular configuration of ACD9 protein outside of mitochondria. I hypothesize that this alternative form of ACD9 has non-enzymatic "moonlighting" functions in the cell. Specific Aim 3 is to elucidate the physiologic function of LCAD. Despite its early recognition, its in vivo metabolic role remains a mystery. Our preliminary data implicates it in bile acid and surfactant metabolism. Specific Aim 3a is to characterize the role of LCAD in bile acid synthesis. I hypothesize that it characterizes a key intermediate step in chenodeoxycholic acid synthesis in a mitochondrial based acidic pathway that is involved in the control of cellular metabolic rate. Specific Aim 3b is to explore the role of LCAD in surfactant metabolism using an LCAD null mouse. These studies necessitate a fundamental revision in our view of mitochondrial 2-oxidation from a metabolic pathway that is only responsible for energy generation to one that is active as well in a variety of previously unrec- ognized functions in other important biologic processes.The acyl-CoA dehydrogenases are important enzymes in maintaining normal chemical balance in the body. We have identified a new genetic disorder of one of these enzymes that leads to liver failure. Studying this disorder is important to learn more about its clinical presentation and treatment.